AbstractIn this unit the preparation and application of enzyme‐nucleotide conjugates is depicted. First, a modified nucleoside triphosphate is synthesized bearing a long and flexible linker equipped with a thiol group. The nucleotide is then reacted with maleimide‐activated horseradish peroxidase to yield an enzyme‐nucleotide conjugate, which due to the long linker, can be used as a substrate by DNA polymerases in primer extension reactions. Finally, an assay based on these findings is described that provides a fast and easy nucleic acid detection and genotyping platform. © 2018 by John Wiley & Sons, Inc.